Abstract Most of our understanding of endosymbiosis originates from the bacterial endosymbionts of multicellular, terrestrial hosts, which represent habitats with dramatically different selective pressures than inside a protist cell. Methanogenic archaea from the genus Methanocorpusculum are among the few known intracellular archaea and form unique symbioses with both animal and protist hosts, providing a unique opportunity to contrast symbiont evolution and function in very distinct host types. Here, we conducted phylo- and pangenomic analyses on 106 Methanocorpusculum strains originating from animal and ciliate hosts as well as environmental habitats. We recovered two divergent clades corresponding to animal gut-associated and intracellular ciliate-associated/environmental lineages and found that ciliate-associated and environmental Methanocorpusculum are virtually indistinguishable functionally and phylogenetically. Ciliate-associated symbionts retained broad biosynthetic capacity and encoded functions related to osmotic stress tolerance and adhesion within the host cell, while animal gut-associated symbionts exhibited patterns of genome streamlining and nutrient scavenging consistent with host supply and immune adaptation. Our findings illuminate how the contrasting selective pressures of protists and animal hosts have driven divergent evolutionary and functional strategies in congeneric archaeal symbionts.

Leontynka is a non-photosynthetic lineage of the order Chlamydomonadales (Chlorophyta). Although many Chlamydomonadales members encode components of the anaerobic energy metabolism, studies focused on Chlamydomonadales algae thriving in hypoxia and not prospering in oxic conditions are missing. Using a combination of experimental approaches, comparative genomics, and advanced in silico protein localization analyses, we employed Leontynka as a model to investigate the evolution of anaerobiosis in Chlamydomonadales. Leontynka spp. accumulate a wide range of storage forms, enabling them to cope with nutritional stresses. Their mitochondria contain well-developed cristae mediating a conventional aerobic energy metabolism. Moreover, colocalization of a Raman signal for cytochromes with the position of mitochondria in the cell indicates that oxidative phosphorylation is an important route of energy metabolism in the alga. Interestingly, Leontynka spp. concentrate enzymes potentially involved in oxygen-independent ATP synthesis within the plastid, which lost the ability to produce ATP using proton gradient generated by membrane complexes that exploit redox reactions. We analyzed the composition of prokaryotic communities co-isolated with Leontynka spp. and hypothesize that their preference for hypoxic/microoxic conditions is facilitated by metabolic interactions with certain microaerophilic and anaerobic bacteria. This study represents the first comprehensive analysis of microaerophilic Chlamydomonadales algae. Having retained several ancestral enzymes of the anaerobic energy metabolism, Leontynka represents a unique vantage point for understanding the evolution of the hydrogen production machinery and adaptations to low oxygen in Chlamydomonadales (and core chlorophytes in general). Our findings suggest that the plastid of non-photosynthetic Leontynka follows a similar evolutionary path as mitochondria when adapting to anaerobiosis and parallels the transition of a mitochondrion into a hydrogenosome.

This study examined the prevalence, epidemiological characteristics, and genotype diversity of Dientamoeba fragilis in a Czech hospital-based cohort. Patients undergoing routine parasitological diagnostics at the Regional Hospital Liberec (Liberec) between 2022 and 2023 formed the primary epidemiological cohort. Additional PCR-positive samples from the University Hospital Bulovka (Prague) collected between 2022 and 2024 were included for molecular characterization. Detection was performed using multiplex real-time PCR (Allplex, Seegene) and routine microscopy. Genotypes were identified by Sanger sequencing of a 674 bp fragment of the SSU rRNA gene, with cloning used to resolve the presence of multiple genotypes within individual samples. Dientamoeba fragilis was detected by PCR in 25.3

Marine anoxic sediments are expansive ecosystems, effectively devoid of oxygen, where eukaryotic life is predominantly represented by protists. In this study, we surveyed a range of such habitats and uncovered novel diversity within ciliated protists from the subclass Scuticociliatia (class Oligohymenophorea). We establish three new genera of marine anaerobic scuticociliates within the family Anaerocyclidiidae that were previously detected exclusively through cultivation-independent environmental surveys. Our results show that marine Anaerocyclidiidae have a global distribution and occur frequently in anoxic sediments. Notably, all studied marine Anaerocyclidiidae host prokaryotic ectosymbionts of varying sizes and shapes, potentially representing distinct prokaryotic lineages. Through broad geographic sampling and the establishment of the largest culture collection of marine anaerobic scuticociliates to date, we investigated the diversity, morphology, behavior, and symbiotic associations within this significant ciliate lineage. These findings highlight the importance of cultivation approaches to uncover novel protistan taxa and their symbiotic relationships, expanding our understanding of biodiversity and ecosystem functioning in oxygen-depleted habitats.

Odontostomatid ciliates, known for over a century, were historically classified within various taxonomic groups of Ciliophora Doflein, 1901 until their reclassification into the class Odontostomatea. Despite the recognition of 25 valid species, most descriptions predate the advent of silver impregnation and sequencing methods. Consequently, many species were described based solely on observations of live specimens, leading to incomplete or ambiguous records. To date, redescriptions of only three species include 18S rRNA gene sequences data, and their evolutionary relationships remain unresolved. In this study, we investigated 32 populations representing 15 species—including three newly described—across the genera Discomorphella, Epalxella, Limnomylestoma gen. nov., Mircalla gen. nov., Mylestoma, Pelodinium, Saprodinium, and Tostonella gen. nov. Comprehensive analyses were conducted using in vivo microscopy, silver impregnation, and scanning electron microscopy. We also designed specific primers to amplify the partial 18S rRNA gene of various odontostomateans and retrieved additional 18S rRNA sequences from environmental metatranscriptomic and metagenomic datasets. This study represents the most extensive investigation of Odontostomatea to date, confirming the monophyly of the class by revealing the position of Epalxella, reconstructing its internal phylogeny, identifying two main odontostomatean lineages, and revealing its remarkable diversity.

Abstract Anaeramoeba pumila is a free-living anaerobic amoeba and the smallest known member of the Anaeramoebae, a phylum characterized by elaborate membrane-bound symbiosomes housing sulfate-reducing bacterial symbionts. Here, we report a draft nuclear genome assembly of A. pumila LANTAAN and describe the discovery, genomic characterization, and metabolic reconstruction of Candidatus Centrionella anaeramoebae gen. nov., sp. nov., an obligate intracellular symbiont of A. pumila belonging to the order Legionellales. Ca. Centrionella is a rare anaerobic member of Legionellales, a lineage otherwise comprising aerobic intracellular pathogens. Its genome (1.52 Mbp, 1,249 genes) is highly reduced and encodes an entirely anaerobic metabolism centered on substrate-level phosphorylation, arginine fermentation, and hydrogen oxidation via a bidirectional [NiFe]-hydrogenase — metabolic strategies that parallel those independently evolved in the distantly related Anoxychlamydiales. The complete Dot/Icm type IVB secretion system is retained and likely mediates ongoing host manipulation, including via a large repertoire of predicted effector proteins. Strikingly, Ca. Centrionella has acquired eukaryotic Rac1-like GTPase genes from its host through horizontal gene transfer, with subsequent domain shuffling and duplication, that it may use to manipulate the cytoskeleton of its host. Unlike other Anaeramoeba symbionts, Centrionella localizes to the host microtubule-organizing center rather than a symbiosome, a localization consistent with cytoskeletal anchoring strategies described in other endosymbionts. The symbiosome, present in other Anaeramoeba species, appears to have been secondarily lost in A. pumila . A co-occurring Desulfobacter sp. LANTAAN, related to symbionts of other Anaeramoebidae, likely forms a tripartite syntrophic consortium by consuming hydrogenosomal fermentation end-products and supplying vitamin B 12 . Together, these findings illuminate the evolutionary transition in Legionellales from aerobic pathogenesis to anaerobic mutualism, providing a new model for the origins of intracellular symbiosis.

Anaerobic single-celled eukaryotes illustrate the remarkable diversity of adaptations to life without oxygen, including multiple mitochondrial reductions and various symbiotic interactions with prokaryotes. However, rare lineages remain virtually unexplored. From 11 collected cells, we conducted an in-depth investigation of the ciliate Legendrea loyezae , a rare predator of microscopic animals. Using single-cell transcriptomics and metagenomics, we generated a high-completeness host transcriptome and macronuclear genome and characterized a novel type of mitochondrion-related organelle. By integrating omics data with microscopy and 3D imaging, we uncover an exceptionally complex endosymbiotic consortium with two methanogenic archaea and two bacteria, including the first strictly anaerobic member of Legionellales , and the most reduced member of Thermodesulfobacteriota , with a 0.5-Mb genome. We infer the metabolic potential and extensive complementarity within this pentapartite system and align it with spatial relationships. Our results expand the known complexity of anaerobic eukaryotes and demonstrate the feasibility of comprehensive analyses of rare protists.

Trachelopyllidae Kent, 1881 is a family of predatory litostomatean ciliates distinguished by a three-rowed dorsal brush composed of two dikinetidal rows and one monokinetid row, and one or more types of organic epicortical scales called lepidosomes. To date, the taxonomy of the nine genera comprising the family has been based, almost exclusively, on the types of scales and their combinations together with cell morphometry. Six of the nine genera are monotypic. To date, molecular data has been available from only four species representing two genera. With the aim of broadening the molecular characterization of the Trachelophyllidae, we present new rRNA cistron sequences of three trachelophyllid species from Idaho, USA, identified by scanning electron microscopy and representing three genera of the family not yet sequenced, and we describe a novel species representing a new genus from the Czech Republic. The results confirm the extraordinarily conserved nature of the 18S rRNA gene and ITS2 regions in the Trachelophyllidae and demonstrate the discordance between lepidosome-based taxonomy and the rRNA cistron-based molecular phylogeny in this group. Additionally, we consider ciliary ultrastructural features unique to members of the Trachelophyllidae and Lingulotrichidae (suborder Trachelophyllina Grain, 1994).

Parabasalid hindgut symbionts of the xylophagous Isoptera/Cryptocercus lineage (Blattodea: Blattoidea) have been attracting the interest of biologists for decades. However, the parabasalids from other cockroaches (Blattodea excluding Isoptera) remain largely unstudied. The genus Hypotrichomonas, along with genus Trichomitus, belongs to the small parabasalid order Hypotrichomonadida. So far, ten valid Hypotrichomonas species have been described from animals, both vertebrates and invertebrates. Here we have uncovered an extensive diversity of this genus in cockroaches. We have obtained 72 isolates from 41 cockroach species and one from a water-filled tree hole. Using a combination of light-microscopic morphology of protargol-stained cells, scanning electron microscopy, and a phylogenetic analysis of SSU rRNA gene, we show that our strains represent multiple novel Hypotrichomonas species, and we formally describe seven of them. Our data show that the evolutionary center of the genus likely lies in cockroaches. Moreover, the new evolutionary lineages of Hypotrichomonas from cockroaches show a high degree of host specificity.

Symbiotic interactions are an ecologically and evolutionary significant phenomenon pertaining to virtually every organism on Earth. For eukaryotes inhabiting extreme environments, syntrophic symbioses with microbes may be key to successfully colonizing new niches, such as globally expanding oxygen-depleted habitats. Multi-domain symbioses between microbial eukaryotes and intracellular methanogenic archaea are crucial to understanding the origins and mechanisms of eukaryotic anaerobiosis. Nearly all anaerobic ciliates, ecologically important protists found in diverse oxygen-depleted environments, host methanogenic endosymbionts, sometimes alongside bacterial partners, that facilitate their anaerobic metabolism. Although vertical symbiont transmission necessarily occurs during ciliate cell division, symbionts might occasionally be acquired horizontally. However, patterns of host-symbiont specificity and intraspecific variability remain poorly understood. Here, we present the first intra-specific genomic analysis of both host and symbionts in such partnerships, providing key insights into the fidelity of eukaryotic-prokaryotic liaisons in anoxia. We assessed the symbiont-host co-diversification and genetic variation across eleven populations of a single undescribed Metopus species hosting Methanocorpusculum cultured from intertidal sediment locations separated by meters to 1000s of kilometers. Our results show incongruency in host mitochondrial and symbiont phylogenies, indicating a mixed transmission mode. On a genomic level, both host and symbiont populations formed distinct location-specific clusters exhibiting no signs of isolation-by-distance. Instead, ecological factors appear to have driven population genomic divergence at least partly and likely led to differences in metabolic traits. Symbiont comparative and population genomics enable us to further comprehend the complex nature of these multi-partner syntrophic symbioses, crucial to interpreting cell-cell interactions across the domains of life. ### Competing Interest Statement The authors have declared no competing interest. Simons Foundation, Early Career Investigator in Marine Microbial Ecology and Evolution Czech Science Foundation, https://ror.org/01pv73b02, 23-06004S

Ciliated protists (Ciliophora) are important members of freshwater, brackish, marine, and hypersaline benthic microbial communities. As part of our broader studies of anaerobic protists, we encountered a ciliate in hypoxic sediment samples from three geographically distant saline habitats and identified it as Spirorhynchus verrucosusCunha, 1915. This highly unusual ciliate has a complicated nomenclatural and taxonomic history and has been assigned to the armophorean family Metopidae. The distinctive cell shape and unique arrangement of ectosymbionts allows identification of this species by in vivo observation. Two populations of another ciliate, found in deep-sea sediments, were recognized as a morphologically and genetically distinct but not yet formally described Spirorhynchus species. Muranotrichea Rotterová et al., 2020 is a recently established class of obligately anaerobic marine ciliates that bear prokaryotic ectosymbionts, and includes one family with two genera and three species. Phylogenetic analyses, based on the first 18S rRNA gene sequences from the genus, place Spirorhynchus in Class Muranotrichea. In single-gene trees, Spirorhynchus is monophyletic, branching as the closest relative of a marine environmental sequence and the muranotrichean genus Thigmothrix, with which Spirorhynchus also shares some morphologic similarities. Here, we also provide the first protargol impregnations and scanning electron microscopy images from Spirorhynchus.

During the past decade, our understanding of eukaryotic evolution has increased immensely. Newly recognized eukaryotic supergroups have been established1-3, and most enigmatic orphan lineages have had their relationships resolved4-6. Studies on unicellular protist eukaryotes have also been key to understanding the evolution of mitochondria, the fundamental organelles of the eukaryotic cell, which originated from an alphaproteobacterial ancestor. The retention of ancestral alphaproteobacterial pathways in some protist lineages reveals that the mitochondrion of the last eukaryotic common ancestor was more metabolically versatile than are the highly derived mitochondria that are found in most modern eukaryotes7,8. Here we report the discovery of such a unicellular eukaryote, Solarion arienae gen. et sp. nov., an inconspicuous, free-living heterotrophic protist with two morphologically distinct cell types and a novel type of predatory extrusome. We assign Solarion to the new phylum Caelestes. Together with Provora, hemimastigophoreans and Meteora, they form a new eukaryotic supergroup, Disparia. Moreover, S. arienae has some noteworthy traits associated with the mitochondrial genome; in particular, the mitochondrially encoded secA gene, a remnant of an ancestral alphaproteobacterial protein secretion pathway, which has been lost almost entirely in extant mitochondria9,10. The discovery of S. arienae broadens our understanding of early eukaryotic evolution and facilitates the study of proto-mitochondrial metabolic remnants, shedding light on the complexity of ancestral eukaryotic life.

The ciliate genus Malacophrys has been incertae sedis for more than 50 years in what is now subphylum Intramacronucleata, provisionally assigned to three different classes by various authors. Of the three species included in the genus, M. sphagni and the type species, Malacophrys rotans, have not been studied by modern methods and M. viridis, although morphologically well-described, lacks molecular characterization. We identified two freshwater ciliates as morphologically consistent with two members of Malacophrys, namely M. rotans and M. viridis. We studied one population of M. rotans, using in vivo observation and silver carbonate and silver nitrate impregnation, and two populations of M. viridis, using in vivo observation, silver carbonate impregnation, and 18S rRNA gene sequencing. Phylogenetic analyses strongly support the position of M. viridis in the class Oligohymenophorea. Morphologic features suggest Malacophrys rotans belongs to order Tetrahymenida, but a confident phylogenetic assignment awaits its molecular sequencing. Differences between their oral structures and silverline patterns indicate that M. rotans and M. viridis likely belong to different genera. A formal separation at the genus level awaits molecular characterization of the type species, M. rotans.

Avian trypanosomes (Trypanosoma, Kinetoplastea) are successful blood parasites occurring worldwide. These parasites are usually non-pathogenic to their avian hosts, thus neglected in studies regarding their life cycles and vectors. Several families of blood-sucking dipteran insects, including mosquitoes, have been identified as vectors of avian trypanosomes. Mosquitoes have been experimentally confirmed as vectors of Trypanosoma culicavium and Trypanosoma thomasbancrofti. In this study, we describe a third species of avian trypanosomes occurring in mosquitoes, designated as Trypanosoma tertium n. sp. This species can be distinguished from related trypanosome species based on morphology and small subunit rRNA gene sequence. Two isolates of T. tertium n. sp. obtained from a mosquito and a bird host were able to infect two subspecies of laboratory Culex pipiens mosquitoes, with infection rates reaching 60% and heavy infections in 90% of positive females. In infected mosquitoes, trypanosomes occurred as long epimastigotes in the midgut and short epimastigotes and rosettes in the hindgut. Putative infectious stages were detected in the diuretic liquid of infected mosquitoes, suggesting, besides transmission through ingestion of the infected vector, a possible transconjunctival infection. Among wild mosquitoes, avian trypanosomes were detected exclusively in Cx. pipiens with 3.3% total prevalence, while T. tertium n. sp. prevalence was only 0.08% among 1128 dissected Cx. pipiens individuals. In birds, T. tertium n. sp. was detected in 8 species within which the prevalence was 1.3% (686 birds), while it was 0.3% in total (3084 birds). We discuss the relationship of the newly described T. tertium n. sp. with other mosquito-transmitted trypanosomes.

The phylum Heterolobosea Page and Blanton, 1985 is a group of eukaryotes that contains heterotrophic flagellates, amoebae, and amoeboflagellates, including the infamous brain-eating amoeba Naegleria fowleri. In this study, we investigate the deep evolutionary history of Heterolobosea by generating and analyzing transcriptome data from 16 diverse isolates and combine this with previously published data in a comprehensive phylogenomic analysis. This dataset has representation of all but one of the major lineages classified here as orders. Our phylogenomic analyses recovered a robustly supported phylogeny of Heterolobosea providing a phylogenetic framework for understanding their evolutionary history. Based on the newly recovered relationships, we revised the classification of Heterolobosea to the family level. We describe two new classes (Eutetramitea cl. nov. and Selenaionea cl. nov) and one new order (Naegleriida ord. nov.), and provide a new delimitation of the largest family of Heterolobosea, Vahlkampfiidae Jollos, 1917. Unexpectedly, we unveiled the first two cases of genetic code alterations in the group: UAG as a glutamine codon in the nuclear genome of Dactylomonas venusta and UGA encoding tryptophan in the mitochondrial genome of Neovahlkampfia damariscottae. In addition, analysis of the genome of the latter species confirmed its inability to make flagella, whereas we identified hallmark flagellum-specific genes in most other heteroloboseans not previously observed to form flagellates, suggesting that the loss of flagella in Heterolobosea is much rarer than generally thought. Finally, we define the first autapomorphy of the subphylum Pharyngomonada, represented by a fusion of two key genes for peroxisomal β-oxidation enzymes.

Ciliates are a diverse group of protists known for their ability to establish various partnerships and thrive in a wide variety of oxygen-depleted environments. Most anaerobic ciliates harbor methanogens, one of the few known archaea living intracellularly. These methanogens increase the metabolic efficiency of host fermentation via syntrophic use of host end-product in methanogenesis. Despite the ubiquity of these symbioses in anoxic habitats, patterns of symbiont specificity and fidelity are not well known. We surveyed two unrelated, commonly found groups of anaerobic ciliates, the Plagiopylea and Metopida, isolated from anoxic marine sediments. We sequenced host 18S rRNA and symbiont 16S rRNA marker genes as well as the symbiont internal transcribed spacer region from our cultured ciliates to identify hosts and their associated methanogenic symbionts. We found that marine ciliates from both of these co-occurring, divergent groups harbor closely related yet distinct intracellular archaea within the Methanocorpusculum genus. The symbionts appear to be stable at the host species level, but at higher taxonomic levels, there is evidence that symbiont replacements have occurred. Gaining insight into this unique association will deepen our understanding of the complex transmission modes of marine microbial symbionts, and the mutualistic microbial interactions occurring across domains of life.

The relationships of the mainly free living, obligately anaerobic ciliated protists belonging to order Metopida continue to be clarified and now comprise three families: Metopidae, Tropidoatractidae, and Apometopidae. The most species-rich genus of the Metopidae, Metopus has undergone considerable subdivision into new genera in recent years as more taxa are characterized by modern morphologic and molecular methods. The genus, Castula, was established to accommodate setae-bearing species previously assigned to Metopus: C. setosa and C. fusca, and one new species, C. flexibilis. Another new species, C. specialis, has been added since. Here we redescribe another species previously included in Metopus, using morphologic and molecular methods, and transfer it to Castula as C. strelkowi n. comb. (original combination Metopus strelkowi). We also reassess the monotypic genus, Pileometopus, which nests within the strongly supported Castula clade in 18S rRNA gene trees and conclude that it represents a morphologically divergent species of Castula.

Psalteriomonadidae are a small family of anaerobic free-living protists belonging to Heterolobosea, Discoba. We cultured 74 new strains of mostly amoeboid Psalteriomonadidae obtained from mainly freshwater habitats and sequenced their 18S rRNA gene. Based on the phylogenetic analysis and genetic distances, we report multiple novel species, four of which we formally describe based on the light-microscopic morphology (Psalteriomonas minuta, P. australis, P. fimbriata, and P. parva). We also examined the ultrastructure of two Psalteriomonas species using transmission electron microscopy. We transfer Sawyeria marylandensis into the genus Psalteriomonas and synonymize Sawyeria with Psalteriomonas. In addition, we studied the flagellate stage of P. marylandensis comb. nov. for the first time, using light and scanning electron microscopy.

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e. endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, although our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments.

The phylogenetic and taxonomic affinities of lineages currently assigned to the non-monophyletic ciliate order Loxocephalida Jankowski (1980) within subclass Scuticociliatia Small (1967) remain unresolved. In the current study, we redescribe the morphology of the type species, Loxocephalus luridus Eberhard (1862) based on two Czech populations and include the first scanning and transmission electron microscopy images of the species. We provide the first 18S rRNA gene sequences for L. luridus and consider its phylogenetic position. Our results support the separation of Dexiotricha from Loxocephalus; however, the former genus is recovered as non-monophyletic. The monophyly of genus Dexiotricha and that of Loxocephalus + Dexiotricha is rejected. Loxocephalus luridus, together with Dexiotricha species, nests within a fully supported clade with Conchophthirus species, long presumed to belong to the Pleuronematida. Haptophrya is recovered as sister to this clade. The monophyly of the Astomatia Schewiakoff (1896) including Haptophrya is rejected. No clear morphologic synapomorphy is identified for the fully supported clade consisting of Haptophrya, Dexiotricha, Loxocephalus, and Conchophthirus.